Requirements for the identification and measurement of biological or chemical entities in a sample typically include a means of isolating or separating the analyte in question. Once spatially, temporally, or otherwise isolated from the surroundings, the analyte provides or promotes a signal whose intensity can be interpreted as a measure of the presence and/or concentration of analyte originally present in the sample. For example, one common approach to enzyme-linked immunosorbant assays (ELISAs) utilizes capture ligands to retain the analyte in question at the bottom of a microtiter plate while irrelevant molecules are removed from the well in a series of washes. When a fluorescently-labelled antibody targeting a second epitope is introduced, the signal from adherent fluorophores can be interpreted as proportional to the concentration of analyte present in the original sample. In this and other embodiments, absolute measurements are achieved via comparison with a parallel signal arising from a known quantity of analyte.
Surface plasmon resonance (SPR) assays are based on the coupling of P-polarized incident light to a surface plasmon wave, a physical mode created by charge density oscillations at a metal-dielectric interface. The mismatch in dielectric constants between these two materials supports a mode of evanescent electron excitation known as a surface plasmon. Coupling of the light to this mode, known as resonance, requires a momentum match between the plasmon and the incident light, and consequently is exquisitely sensitive to the properties of the light. When conditions are optimal, the majority (>90%) of the light acts to excite the plasmon, with little being reflected. Detectors placed in the optical path of reflected light are able to measure this intensity as a function of incident angle, wavelength, or phase, and instrumentation has been developed that generates intensity plots as a function of each of these variables. The nadir of the intensity plot is known as the SPR minimum, and it defines the conditions where optimal coupling into the plasmon mode occurs.
The value of the independent variable defining the SPR minimum changes with the dielectric constant at the surface, a phenomenon which underlies the utility of SPR techniques in biological or chemical sensing. Accumulating mass (e.g. protein binding) at the sensor surface leads to a proportional change in the dielectric constant, and a consequent change in the value associated with the SPR minimum. This “SPR shift” acts in a sense like a “molecular scale,” providing quantitative measures of changes in mass at the sensor surface. Thus SPR acts in a sense like a “molecular scale,” where change in surface mass recorded as an “SPR shift.” Detection of a specific biological molecule can occur with the attachment of an appropriate capture ligand to the sensor surface, (antibodies, oligonucleotides, aptamers, etc.) serving to capture the analyte of interest present in a sample as it flows over the surface. Additional flow of buffer provides a wash sequence to remove non-specific adherents. While this approach has much in common with ELISA, a secondary antibody is not required to measure the SPR signal; the accumulation of mass is theoretically sufficient. Alas, SPR has not proven sensitive enough to detect trace quantities of small molecules, particularly in complex media, and the low sensitivity of classic approaches to SPR as compared to labeled techniques like ELISA has proven to be a limitation to the application of this technology to environmental and clinical samples.
SPR is an evanescent phenomenon and therefore the effect on the dielectric constant is limited to the region immediately surrounding the mass. Indeed in at least one instrument architecture, densities exceeding 1,000 spots/cm2 have been achieved with no significant cross-talk. This opportunity for spatial separation of multiple analytes utilizing an assortment of capture ligands permits an increase in the number of experiments performed on a single chip. When spotted in a 2D array, parallel intensity measurements can be performed using a camera, with pixel clusters defined in silico around each antibody spot. These regions of interest (ROI) serve as independent assays of analytes present in the sample, with shifts in the SPR minima of each ROI providing binding kinetics and end-point concentrations.
The Kretschmann Configuration—The most common method for momentum matching between incident light and the surface plasmon mode is known as the Kretschmann configuration and is dependent on the sensor surface being in optical contact with a high-refractive index prism. Limitations of the Kretschmann configuration include instrument size, instrument price, ease of use, and the relatively small number of simultaneous analyses that can be performed. The physics of this configuration as an SPR detection scheme is well-established (see Homola, J., 2006, “Surface Plasmon Resonance Based Sensors”, Springer Series on Chemical Sensors and Biosensors: Methods and Applications: 1-252), but for the purpose of this disclosure one key element of the design should be noted. Kretschmann instrumentation architecture most frequently contains a single metal layer with two pertinent surface interfaces, to be known going forward as the metal-sample interface and the metal-prism interface. In this configuration, coupling is dependent not only on the dielectric constants of the metal and the sample, but also on that of the prism as can be seen in equation 1:
                              tan          ⁡                      (                                          κ                sp                            ⁢                              d                /                2                                      )                          =                                                            γ                1                            ⁢                                                ɛ                  2                                /                                  κɛ                  1                                                      +                                          γ                3                            ⁢                                                ɛ                  2                                /                                  κɛ                  3                                                                          1            -                                          (                                                      γ                    1                                    ⁢                                                            ɛ                      2                                        /                                          κɛ                      1                                                                      )                            ⁢                              (                                                      γ                    3                                    ⁢                                                            ɛ                      2                                        /                                          κɛ                      3                                                                      )                                                                        (        1        )            where κsp is momentum of the plasmon wave that exists on a dielectric-metal-dielectric and κ2=ω2∈2∈0μ0−β2 and γ1,32=β2−ω2∈1,3∈0μ0. d is the thickness of the metal layer, ω is the angular frequency, ∈0 is permittivity of free space and ∈n is the dielectric constant of medium n, μ0 is the free-space permeability, and β is the propagation constant of the plasmon mode. In the Kretschmann configuration, ∈1 is the dielectric constant of the sample, ∈2 is that of the metal, and ∈3 is that of the prism. The metal layer is made sufficiently thin (˜50 nm) that changes in the resonant conditions established by sample accumulation at the metal-sample interface are affected by the dielectric constant of the prism at the metal-prism interface. This reduced thickness permits the plasmon to “penetrate” into the prism, and allow for coupling conditions to be interrogated from the opposite side of the metal film as the sample.
Grating-coupled SPR (GCSPR)—This approach achieves momentum matching using diffracted light, often produced by means of a sensor chip with an embossed diffraction grating. This coupling scheme simplifies sample preparation, reduces instrument cost, and allows for epi-illumination optics, vastly increasing the number of assays performed simultaneously. Although detection of the SPR minimum in a GCSPR system can be readily achieved by varying angle, wavelength, or phase, this discussion will focus on an angle-scanning approach to GCSPR. Similar principles apply for these other detection modalities, but an angle-scanning approach simplifies equations and facilitates direct comparison with commercially available technologies. In these platforms, much like their Kretschmann counterparts, changes in dielectric constant due to bound mass (Δ∈1) affect κsp, while the values of ∈2 (the metal) and ∈3 (the underlying substrate) do not vary. Similarly, in GCSPR, the change in κsp is detected by varying the incident angle (θ) until the light's momentum (k-vector) matches κsp, and resonance is achieved. The equation that governs coupling into the GCSPR system is reproduced in equation 2:κsource sin θ+mκgrating=κsp(∈1,∈2,∈3)  (2)The above shows that when κsource and κgrating are fixed, as occurs readily in classic GCSPR instruments, the mass-dependent changes in ∈1 shape κsp and are observed as changes in the coupling angle (θ). Local refractive index changes at the metal-dielectric interface are detected with incident light that passes through this dielectric, so in this configuration, the thickness of the metal is irrelevant, as long as it exceeds the minimum necessary to support SPR. Due to the robustness and the relative ease of manufacture, metal layers in most GCSPR platforms have been sufficiently thick (˜1 μm) as to obscure any effect of ∈3 on the plasmon coupling conditions. While GCSPR systems offer considerable improvement over the limitations of Kretschmann systems, they remain less sensitive than fluorescent techniques, and the dependence on moving parts for angle scanning makes these instruments relatively fragile and therefore ill-suited for field use.
Surface Plasmon-Coupled Emission (SPCE)—It has been observed that energy from surface plasmons can be out-coupled and absorbed by fluorophore molecules in close proximity to the metal surface (see Lackowicz, J. R., 2006, “Plasmonics in Biology and Plasmon-Controlled Fluorescence”, DOI 10.1007/s 11468-005-9002-3). The local field of the propagating wave at the metal/dielectric boundary enhances absorption of plasmons as compared to free-space absorption. The subsequent fluorescent emission is out-coupled into propagating lobes in accordance with the momentum matching conditions previously described. Fluorescence generated in this manner is emitted as directional lobes rather than omnidirectionally as in a solution (i.e., as in a typical fluorimeter). An optical detection system can be designed to capture this SPCE with much greater efficiency than can be done with omnidirectional fluorescence. This enhanced capture efficiency results in considerably greater detection sensitivity and is sufficient to quantitatively measure cytokine secretion from single cells (see Reilly, M. T., et al., 2005, “SPR surface enhanced fluorescence with a gold-coated corrugated sensor chip” Progress in Biomedical Optics and Imaging—Proceedings of SPIE Volume 6099, Article number 60990E DOI: 10.1117/12.646165).
Electro-optic polymers are polymers with non-linear optical properties, such that they change their dielectric constant and refractive index as a function of an applied electric field. Polymers displaying the Pockel's or Kerr effect in response to applied voltage have been described and are in use in high speed optical switches in the telecommunications industry. The considerable thermal, chemical, and temporal stability are a sine qua non for commercial optical switches, and this patent aims to take advantage of recent advances enabling nanoscale patterning of said polymers.